Real-time RT–PCR correlates with immunocytochemistry for the detection of disseminated epithelial cells in bone marrow aspirates of patients with breast cancer

Abstract

Real-time reverse transcriptase -polymerase chain reaction (RT -PCR) is a technique with the potential of improving the quantification of disseminated epithelial cells (DEC) in haematological tissues due to its exquisite sensitivity. This sensitivity may lead to false positivity. Immunocytochemistry (ICC) is regarded as the standard methodology to diagnose DEC. In this study, detection with ICC was compared with quantitative real-time RT -PCR for CK-19 and mammaglobin (hMAM) mRNA in bone marrow (BM) of patients with metastatic breast cancer (MBC). Bone marrow was aspirated from 14 control patients and from 29 patients with MBC. Mononuclear cells (MNC) were isolated. Immunostaining was carried out with the Epimet kit. Quantitative PCR was performed on the ABI Prism 7700. The CK-19 and hMAM mRNA quantities were normalised against b-Actin and calculated relative to a calibrator sample (relative gene expression). All controls were negative by ICC and for hMAM expression measured by RT -PCR, whereas the median RGE value for CK-19 was 0.57. For the MBC patients, the median RGE for hMAM was 0 and 10 out of 25 (40%) tested positive. Median RGE for CK-19 was 2.9 and 20 out of 25 (80%) tested positive. With ICC, the median value was 1 stained cell per sample, and 15 out of 24 (62%) samples were positive. A correlation was observed between CK-19 and hMAM expression (r ¼ 0.7; P ¼ 0.0003), and between hMAM expression and ICC (r ¼ 0.6; P ¼ 0.003). CK-19 expression and ICC (r ¼ 0.9; Po0.0001) showed the strongest correlation. Reverse transcriptase -polymerase chain reaction for CK-19 resulted in a higher number of positive BM samples of patients with MBC than ICC. Since an excellent correlation is observed between ICC and RT -PCR, and RT -PCR is probably more sensitive with the advantage of being less observer dependent and thus also more easy to automate, we consider our quantitative real-time RT -PCR method as validated for the detection of DEC in the bone marrow of breast cancer patients.

Expression of membrane type-1 matrix metalloproteinase, MT1-MMP in human breast cancer and its impact on invasiveness of breast cancer cells

“…This relatively stable cytoskeletal associated protein has been widely used as a marker of cancer cells in tissues other than mammary gland, such as lymph nodes and bone marrow and has been valuable in identifying the metastatic cells in these tissues (35,36). The marker is not only useful in identifying cancer cells, it has been recognised as an epithelial marker.…”

Osteopontin‐c is a selective marker of breast cancer

“…The primer sets for osteopontin (OPN) were designed to distinguish the splice variants. The primer sets for CK-19, 15 b-actin and aP-2 covered two different exons to avoid amplification of any contaminating genomic DNA. Amplification efficiencies were determined for each given primer set by cDNA dose-response curve analysis.…”

Circulating tumour cell detection: a direct comparison between the CellSearch System, the AdnaTest and CK-19/mammaglobin RT–PCR in patients with metastatic breast cancer

“…Immunocytochemical analysis is usually used in combination with density-gradient centrifugation (Balic et al, 2005;Muller et al, 2005;Wiedswang et al, 2006), size filtration (Kahn et al, 2004;Wong et al, 2006) or flow cytometry (Meng et al, 2004;Allan et al, 2005) to enrich tumour cells before their detection. In addition, nucleic-acid-based approaches for cell detection have been described (Lambrechts et al, 1999;Smith et al, 2000;Aerts et al, 2001;Stathopoulou et al, 2002;Benoy et al, 2004). There is, however, substantial variability with regard to the rates of positive samples using existing techniques.…”

“…This assay has been previously developed in our laboratory for the detection of CK-19 and mammaglobin transcripts in peripheral blood and bone marrow samples of patients with breast cancer (Benoy et al, 2004). CK-19 is expressed in the majority of breast carcinomas (Bartek et al, 1985) and has been extensively used as a marker for CTC (Slade et al, 1999;Smith et al, 2000;Aerts et al, 2001;Stathopoulou et al, 2003;Benoy et al, 2004;Ring et al, 2005). The specificity of mammaglobin for the detection of breast cancer cells in haematopoietic products has been evaluated in several studies (Leygue et al, 1999;Zach et al, 1999;Suchy et al, 2000;Corradini et al, 2001;Silva et al, 2002;Lin et al, 2003).…”

“…This assay has been previously developed in our laboratory for the detection of CK-19 and mammaglobin transcripts in peripheral blood and bone marrow samples of patients with breast cancer (Benoy et al, 2004). CK-19 is expressed in the majority of breast carcinomas (Bartek et al, 1985) and has been extensively used as a marker for CTC (Slade et al, 1999;Smith et al, 2000;Aerts et al, 2001;Stathopoulou et al, 2003;Benoy et al, 2004;Ring et al, 2005).…”